Reducing medium for the cultivation of Porphyromonas gingivalis.

Porphyromonas gingivalis, a micro-organism frequently associated with human periodontal diseases, is commonly cultured under a reducing atmosphere in enclosed cabinets or glove boxes. This paper reports a modified Wilkins-Chalgren (MWC) medium for the culture of P. gingivalis under atmospheric conditions at 37 degrees C. On the basis of preliminary tests, WC broth was supplemented as follows: 500 mg/l cysteine hydrochloride; 250 mg/l sodium thioglycolate; and 1,000 mg/l sodium bicarbonate. Three P. gingivalis isolates (JKG-I, 33277 and A7436) showed very similar growth over 24 and 48 h periods when cultured both in WC medium in an anaerobic chamber and in the MWC medium. Culture of these isolates both in the anaerobic chamber and in the MWC medium yielded very similar data with respect to trypsin-like activity, total protease activity, and reactivity to monoclonal antibodies specific for P. gingivalis. Growth in the MWC medium varied over a 3- to 4-fold range for seven additional isolates (JKG-7, D86B6, D13B11, D84D2, JKG9, D67D9, D82F5) over 24 and 48 h periods.

Salivary levels of alpha 2-macroglobulin, alpha 1-antitrypsin, C-reactive protein, cathepsin G and elastase in humans with or without destructive periodontal disease.

Five host-response indicators were measured by enzyme-linked immunosorbent assays on unstimulated whole saliva samples from 45 adults (19 male, 26 female). The participants were distributed among four dentate groups representing oral health (I), gingivitis (II), moderate periodontitis (III), and severe periodontitis (IV), and one group of edentulous volunteers (V). Levels of the host-response indicators varied widely, from zero, primarily with groups I and V, to relatively high values with groups II, III and IV. The levels ranged as follows: alpha 2-macroglobulin, 0-4941 ng/ml; alpha 1-antitrypsin, 2-2271 ng/ml; C-reactive protein, 0-472 pg/ml; cathepsin G, 0-6035 ng/ml; elastase, 0-164 ng/ml (free), 0-732 ng/ml (bound to alpha 1-antitrypsin), and 0-318 ng/ml (bound to alpha 2-macroglobulin). Statistical evaluation by planned contrasts showed that levels of host-response indicators for group I were significantly lower (except for alpha 1-antitrypsin) than for groups II, III, and IV. A trend analysis of groups I-IV showed that mean scores (again, except for alpha 1-antitrypsin) increased significantly in a positive, monotonic manner. Group V showed significantly lower values for elastase than in the other groups. The findings demonstrate that these factors can be detected in whole saliva and suggest that, except for alpha 1-antitrypsin, their levels are directly related to an individual's periodontal status.

Evaluating chemical inactivation of viral agents in handpiece splatter.

The water spray used with a modern high-speed dental drill is a significant vehicle for dispersion of infectious agents into the environment, putting patients and the dental staff at risk of infection. This study examines whether disinfectants added to the handpiece water supply could inactivate viral contaminants in splatter. Results supporting use of ethanol or sodium hypochlorite are presented.

Comparison of human polymorphonuclear neutrophil elastase, polymorphonuclear neutrophil cathepsin-G, and alpha 2-macroglobulin levels in healthy and inflamed dental pulps.

Polymorphonuclear neutrophils (PMNs) are found in dental pulp secondary to carious exposures, periodontal disease, or trauma. Lysosomal degranulation of these cells liberates cellular proteases, including elastase (PMN-E) and cathepsin-G (PMN-CG), which produce connective tissue degradation. However, nonspecific pulpal tissue destruction can be modified by a naturally occurring serum protease inhibitor alpha 2-macroglobulin (A2-M). This study relates the concentrations of human PMN-E, PMN-CG, and A2-M in healthy and inflamed pulpal samples. Evaluation of 21 specimens yielded statistically significant differences between healthy and moderate to severely inflamed pulps for all groups (p < 0.05). No significant correlation was detected among human PMN-E, PMN-CG, and A2-M in the healthy tissues (P > 0.05). However, in the moderate to severely inflamed pulps, there was a significant correlation between PMN-CG and A2-M (p < 0.05).

Trypsin-like activity levels of Treponema denticola and Porphyromonas gingivalis in adults with periodontitis.

Treponema denticola (Td) and Porphyromonas gingivalis (Pg) are associated with human moderate and severe adult periodontal diseases. This study quantifies these two anaerobes and their trypsin-like (TL) activities in subgingival plaque collected from both clinically healthy and periodontally diseased sites of human periodontitis patients. Antigen levels of the microorganisms were determined by monoclonal antibodies and TL activities were measured by the fluorescent substrate Z-gly-gly-arg-AFC in a disc format. Significant positive correlations were observed between the antigen levels and the TL activities when the data were subjected to statistical analyses both on a site-specific and on a patient basis. Anaerobe synergism was found between Td and Pg in a continental US population, and positive correlations were found between anaerobe levels (individually and total) and clinical indicators of adult periodontitis.

Measurements of interleukin-6 in gingival crevicular fluid from adults with destructive periodontal disease.

This paper describes a study of whether or not the amounts of interleukin-6 (IL-6) in gingival crevicular fluid (GCF) are correlated with periodontal clinical measures. A sensitive ELISA was developed to measure IL-6 in GCF. Two male and 3 female adult subjects with periodontal disease were examined at their first appointments, after 3 months, and after 6 months. Data were obtained on plaque index (PI), bleeding index (BI), probing depth (PD), and on the IL-6 content of GCF samples from 16 sites per subject for a total of 240 measurements. Significant correlations were found between BI and IL-6 (P < 0.005) and between PD and IL-6 (P < 0.05), but not between PI and IL-6. Only 6 out of the 80 sites (in 3 of the 5 subjects) showed PD increases of at least 2 mm. However, for each of these 3 subjects, the amounts of IL-6 in the GCF samples from these sites were markedly higher than the mean amounts of IL-6 in the GCF samples from the remaining sites. These findings suggest that IL-6 may be a useful indicator of periodontal disease, although more extensive longitudinal studies are needed to determine the real clinical value of this GCF component.

Transamidase and collagenase activity in healthy and diseased human gingival tissues.

Transamidases are a class of calcium-dependent mammalian enzymes which cross-link proteins by catalyzing the formation of (gamma-glutamyl)-epsilon-lysine bonds. It is possible that these enzymes play an important anabolic role in tissue healing. This study was to quantitate transamidase activity in human gingival tissue and examine the relation between transmidase activity and degree of inflammation. Forty-four out of a total 120 collected human gingival specimens from healthy and diseased patients were selected based on histometric and microbiologic criteria. Specimens were minced and homogenized in 10 mM CaCl2 and then extracted for 30 min, in 50 mM tris-HCl buffer (pH 7.5) containing 100 mM CaCl2. Following low speed centrifugation at 4 degrees C, the supernatant solution was assayed for both transamidase and collagenase activities by radioactive amine incorporation, and digestion of tritiated collagen, respectively. Appreciable levels of transamidase and collagenase activities in healthy gingivae were found. These enzyme activities were significantly elevated in the diseased and healing tissues. Unlike other transamidases, calcium was required in the enzyme extraction process.

A sensitive method for the detection of immune complexes in human gingival crevicular fluid.

An assay for immune complexes [ICs] in gingival crevicular fluid [GCF] could be potentially useful to ascertain the risk of periodontal disease activity at specific oral sites. This paper describes and ELISA-based method sufficiently sensitive to measure ICs in GCF samples at levels below 1 microgram/ml. Static GCF samples taken from 5 adults showed IC levels ranging from 5 to 166 micrograms/ml. Additional tests of 10 GCF samples from 1 adult were conducted with complement components and indicated that either C1q or rabbit anti-human C3d was suitable as a capture agent in the assay procedure. Further application of this assay may help to assess the usefulness of IC levels in GCF samples as possible diagnostic indicators of periodontal disease activity.

Natural modifiers of the inflammatory process in the human dental pulp.

Concentrations of the protease inhibitors alpha 1-antitrypsin and alpha 2-macroglobulin were determined in normal and inflamed human dental pulps. Carious pulpal exposure which is associated with polymorphonuclear leukocyte infiltration and release of lysosomal enzymes was chosen as the point of verifiable inflammatory activity in the pulp. Normal samples were collected from nondiseased third molar teeth treatment planned for extraction and inflamed human pulps were collected from teeth with deep carious lesions. One half of each sample was assayed for concentration of protease inhibitors by enzyme-linked immunosorbent assay and the remaining half was examined histologically to verify the clinical diagnosis and categorize the extent of the inflammatory process. alpha 1-Antitrypsin and alpha 2-macroglobulin were detected in normal and inflamed human dental pulps in the nanogram per milliliter range. Statistically significant differences were found in the concentrations of alpha 2-macroglobulin (p less than 0.01) in moderate to severe inflammation versus normal pulp categories and between mildly inflamed pulps and moderate to severely inflamed pulps (p less than 0.05). Although differences in concentrations of alpha 1-antitrypsin were seen between inflamed and normal pulps, the differences were not statistically significant. The presence of these two protease inhibitors in the human dental pulp tissue and the increase in their concentration in acute inflammation indicates that these proteins play a role in the pathogenesis of pulpal inflammatory disease.

Human polymorphonuclear granule components: relative levels detected by a modified enzyme-linked immunosorbent assay in normal and inflamed dental pulps.

Lysosomal granules of polymorphonuclear leukocytes (PMN's) contain proteolytic enzymes and other components important in the regulation of inflammation and the elimination of bacteria or debris associated with pulp disease. However, PMN lysosomal degranulation is nonspecific and can result in destruction of healthy connective tissue adjacent to the areas of damaged or infected tissue. For this study a modified enzyme-linked immunosorbent assay was used to detect the human PMN lysosomal granule products: elastase, cathepsin G, and lactoferrin. Evaluation of 55 pulp samples yielded a statistically significant difference (p less than 0.05) among the levels of elastase and lactoferrin in normal and moderate to severely inflamed pulps. Although cathepsin G levels were increased, there was no statistical significance (p greater than 0.05) among groups. The results indicate that a modified enzyme-linked immunosorbent assay technique can be used to measure PMN lysosomal granule components in dental pulp tissues. Additionally, elastase and lactoferrin levels appear to be valid diagnostic markers of advanced dental pulp disease.

Determination and relationship of C-reactive protein in human dental pulps and in serum.

C-reactive protein (CRP), an acute phase protein synthesized by the liver, increases in serum as much as 3000 times above its normal level in response to acute inflammation. The purpose of this study was to determine whether CRP levels in dental pulps could be correlated with the histological disease status of the pulp and with systemic blood levels of CRP. Inflamed and necrotic pulps were extirpated during routine endodontic therapy. Normal pulps were removed from extracted, intact third molars. One half of each pulp specimen was placed in formalin for histological study; the other half was frozen for immunological study. A serum sample was obtained from each patient at the end of the dental visit. CRP levels were determined by the enzyme-linked immunosorbent assay. Pulps were categorized histologically as normal, inflamed, inflamed/necrotic, or necrotic. The correlation between CRP levels of pulp and serum was not significant. CRP levels of normal pulps differed significantly only from inflamed pulps (p less than 0.05, Dunnett). This increase in CRP appears to be a local phenomenon resulting from the interaction of CRP with various inflammatory mediators in the pulp.